Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Vaccinia virus, the prototype for this group, has a 180,000 base pair linear double-stranded DNA genome with covalently linked ends and a 10,000 base pair inverted terminal repetition. Nucleotide sequence analysis revealed that the ends of the genome consist of hairpin loops so that the two DNA strands form one continuous polynucleotide chain. The 104 nucleotide apex of the loop is rich in adenosine and thymidine nucleotides, incompletely base-paired and exits in two isomeric forms that are inverted and complementary in sequence (flip-flopped). During replication, head to head concatemers are formed. Models for the replication of the terminal segment of the poxvirus genome that involve site specific nicking and lead to flip-flop rearrangements and head to head dimerization have been proposed. Enzymes involved in DNA replication are encoded within the vaccinia virus genome. Using phosphonoacetate resistance as a genetic marker, the DNA polymerase gene has been precisely mapped. Hybridization of mRNA, cell-free translation, and peptide mapping confirmed the location of the DNA polymerase gene.